Purification and characterization of a new alkali-thermostable lipase from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere

Abstract

An extracellular lipase (EC 3.1.1.3), SAL-PP1, from Staphylococcus aureus isolated from Arachis hypogaea rhizosphere was purified and characterized. The enzyme was purified using PALL'S Microsep centrifugal device (10kD cut off), hydrophobic interaction (phenyl sepharose CL-4B column) and Superose-12 gel filtration chromatography and found to have a molecular mass of around 49kDa. The gene fragment encoding the part of the catalytic site of the SAL-PP1 lipase was sequenced and the deduced amino acid sequence shows 93% identity with that of SEL3. SAL-PP1 showed activity against long acyl-chain triglycerides, various p-nitrophenyl esters and phospholipids. The enzyme shows high stability and activity after incubation with various metal ions (retained >90% activity in presence of Ca2+, Na+, Cu2+, Mg2+, Fe2+, or Hg2+ at 10mM), organic solvents (retained >80% activity in presence of acetonitrile, ethanol, DMSO, methanol, isopropanol, toluene, or ethylene glycol at 10mM), detergents (retained >70% activity in Triton X-100, Tween 80, or sodium deoxycholate at 10mM) and irreversible inhibitors (retained >77% activity in presence of PMSF, leupetin, or β-mercaptoethanol, at 1mM). Thermal inactivation studies revealed a temperature dependent unfolding of secondary structure of protein. SAL-PP1 showed maximal activity and stability at pH 8.0 and pH 9.0, respectively. The alkali-thermostability, organic solvent-tolerance and broad substrate specificity of this enzyme may have potential implications in detergent formulations, biotransformation, industries, and medicine.